Interdepartmental Laboratory of Electron Microscopy, University Roma Tre, Roma, Italy
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Globins are present in all kingdoms of living organisms where they display a variety of functions, including the O2 sensing, transport, and storage, the synthesis and scavenging of reactive nitrogen and oxygen species, and heme-based catalysis (see refs.  and ).
Nerve globins have been observed in nonvertebrates and vertebrates , the first nerve globin having been recognized in the nerve cord of the polychaete annelid Aphrodite aculeata in 1872 . Only in 2000, the first vertebrate nerve globin, named neuroglobin (Ngb), has been identified in neuronal tissues of humans and mice . Recently, cytoglobin (Cygb), hemoglobin (Hb), and myoglobin (Mb) have been reported to be expressed in the mammalian nervous system [6-18]. Phylogenetic analysis of globins expressed in Homo sapiens, Mus musculus, and Rattus norvegicus nervous system indicates, that they have been evolved from a common ancestor, resulting in four well-supported clades, representing Hbs, Mbs, Cygbs, and Ngbs (see Fig. 1 and Supporting Information).
Mammalian nerve globins colocalize not only in the brain and in the neuronal retina but also in different tissues and organs [5-7, 9, 11, 13, 16-36]. The concentration of mammalian nerve globins is generally ∼1 μM [7-12, 16-18, 37]; Ngb has been found at relatively high concentration in neurons of the hypothalamus  reaching high levels (from 100 to 200 μM) in the retina rod cells [19, 22]. The low concentration of mammalian nerve globins suggests that they are not simply involved in O2 storage and supply to mitochondria, but could display (pseudo-)enzymatic actions and could be part of signaling pathways. Remarkably, the coexpression of Ngb, Cygb, Mb, and Hb in the brain suggests that they could play specific functions [7, 8, 10, 14, 16, 26, 33, 38-53]. Here, the putative roles of mammalian nerve globins are reviewed.
In 2000, the third member of the globin family was reported to be expressed in human and mouse brain, and therefore it was named Ngb (17 kDa) . Ngb is expressed not only in neurons of the central and peripheral nervous systems, but also in glioblastoma cell lines, in quiescent astrocytes of the healthy seal brain, in reactive astrocytes in neuropathological models of traumatic injury, infectious, autoimmune, and excitotoxic diseases, in the gastrointestinal tract, and in endocrine organs [5, 13, 16, 17, 20, 26, 29, 30, 32, 33, 49]. Human Ngb occurs at relatively low concentrations in most tissues and organs including resting neurons (∼1 μM), and its concentration has been estimated to be up to 100–200 μM in highly metabolically active cells (e.g., of the retinal ganglion cell layer and of the optic nerve (5, 17, 19, 20, 26, 29, 30, 42, 43, 46, 54, 55).
Members of the Ngb family display a bis-histidyl hexa-coordinated heme-Fe atom , the cleavage of the heme distal HisE7-Fe bond representing a prerequisite for exogenous ligand binding [57-60]. The reversible penta- to hexa-coordination transition of the heme-Fe atom, and in turn human Ngb reactivity, is allosterically controlled by the redox state of the cell and phosphorylation [58, 61]. The cleavage of the CysCD7-CysD5 disulfide bond stabilizes the hexa-coordinated form of human Ngb and in turn reduces the O2 affinity for the ferrous derivative (i.e., P50 increases from 0.9 to 9.2 mm Hg). However, this mechanism does not apply to murine Ngb that displays a Gly residue, instead of Cys, at the CD7 position . Human Ngb phosphorylation at putative sites SerA7, SerA12, SerAB2, SerCD10, SerCD11, and SerDE3 by intracellular kinases (e.g., ERK and PKA) increases the nitrite reductase activity by stabilizing the penta-coordinated derivative of the heme-Fe atom. Of note, binding of the scaffold protein 14-3-3 at putative sites ArgA9-ProAB3 and ArgCD7-ProD1 of human Ngb stabilizes the phosphorylated heme-protein inhibiting dephosphorylation .
Although the correlation between the formation and decomposition of reactive oxygen and nitrogen species and human Ngb in vivo is still elusive [26, 62], some in vitro evidences have been reported. Indeed, human Ngb displays a protective role by scavenging NO in the presence of high O2 levels [63-65]. Nonetheless, at low O2 concentrations, human Ngb acts as a nitrite reductase producing NO, thus regulating intracellular hypoxic NO signaling pathways [66, 67]. Moreover, although human Ngb does not react with H2O2 [41, 63], the human Ngb–NO2 adduct reacts with H2O2 catalyzing the nitration of aromatic substrates .
In cell culture systems, human Ngb expression is moderately induced by hypoxia , H2O2 toxicity , and lipopolysaccharide . These data suggest that human Ngb is a stress-inducible protein that could behave as a compensatory protein responding to injuring stimuli. The human Ngb expression can be enhanced experimentally by cobalt, deferoxamine, hemin, and some short-chain fatty acids, valproic and cinnamic acids . Human Ngb levels are also enhanced by HIF1α , although apparently indirectly, and by treatment with the vascular endothelial growth factor (VEGF; ref. ).
Remarkably, Ngbs cope with cerebral hypoxia in diving mammals by either facilitating oxygen supply or protecting from reactive oxygen species. However, Ngb mRNA expression levels were 4–15 times higher in the brains of harbor porpoises and minke whales than in terrestrial mammals or in seals. This indicates that different strategies in seals and whales resulted from a divergent evolution and an independent adaptation to diving .
Recently, 17β-estradiol (E2) has been shown to upregulate Ngb levels in a human neuroblastoma cell line, in mouse primary hippocampal neurons, and in mouse primary astrocytes [46, 48]. The E2-induced human Ngb upregulation plays a pivotal role in the neuroprotective effect of this hormone against H2O2-induced apoptosis in neurons  and for the anti-inflammatory mechanisms of E2 in astrocytes . Thus, Ngb should also be regarded as a hormone-inducible protein whose upregulation could protect neurons against death induced by injuring stimuli .
Human Ngb interacts with several proteins , including those involved in ionic homeostasis maintenance, energy metabolism, mitochondria function, and signaling pathways for cell survival and proliferation [26, 33, 46]. Among others, the Gα protein, the voltage-dependent anion channel (VDAC; a critical regulator of mitochondria permeability transition pore opening), a subunit of mitochondrial complex III, cytochrome c1, and a subunit of the mitochondrial complex III (critical for mitochondrial ATP production and the generation of superoxide anion) have been identified [33, 61]. In particular, Gα protein binding to NGB inhibits GDP dissociation, thereby protecting neuronal cells against oxidative stress [72, 73]. Moreover, human cytosolic Ngb binding to VDAC may inhibit mitochondria permeability transition pore opening after O2 or glucose deprivation, block cytochrome c release from mitochondria, and impair the subsequent apoptosis . On the other hand, recent data indicate that both E2 stimulation and H2O2 challenge induce a reallocation of human Ngb from nucleus to mitochondria . After E2 treatment and the H2O2 insult, the reallocated human Ngb associates with cytochrome c in mitochondria sustaining the idea that human Ngb could prevent stress signals inducing cytochrome c release into the cytosol. However, only E2 stimulation induces the expression of human Ngb facilitating the association to cytochrome c into mitochondria and reducing the amount of cytosolic cytochrome c. Interestingly, it has been speculated that the interaction of human Ngb with cytochrome c in mitochondria [30, 51, 74] outlines the Ngb capability to reduce cytochrome c suggesting an important protective role against programmed cell death [40, 42, 45]. Moreover, the activation of caspase-3 is very sensitive to the human Ngb/cytochrome c ratio activating the intrinsic pathway of apoptosis . In the presence of the 1/1 human Ngb/cytochrome c ratio, human Ngb is unable to impair the activation of caspase-3 and consequently to protect cells from apoptosis. Cell protection has been postulated to be achieved when the human Ngb/cytochrome c ratio is at least 3/1, indicating that the human Ngb/cytochrome c ratio may represent the “trigger level” that determines the cell fate .
In vivo experiments, using transgenic rodents, have shown that increased levels of Ngb significantly protect both heart and brain tissues from hypoxic/ischemic and oxidative stress-related insults, whereas decreased Ngb levels lead to an exacerbation of tissue injury [73, 75-77]. Moreover, increased levels of human Ngb protect neurons from neurodegenerative disorders such as Alzheimer's disease [33, 51]. Of note, human Ngb has been reported to interact specifically with the prion protein . Moreover, human Ngb overexpression attenuates tau hyperphosphorylation at multiple Alzheimer's disease-related sites . However, the overexpression of human Ngb is necessary for neuroprotection [26, 33, 40, 42]. In line with this idea, overexpression of human Ngb confers protection to cultured neurons against hypoxia [72, 80, 81], anoxia, and glucose deprivation [21, 82]. Moreover, human Ngb overexpression is protective against models of neurological disorders including Alzheimer's disease .
As a whole, these data indicate that human Ngb overexpression could protect neurons against death induced by oxidative stress, mitochondrial pathologies, and neurotoxicity. These findings open a new scenario for human Ngb in neuroprotective antiapoptotic mechanisms in which different human Ngb pools, possessing a diverse intracellular localization, synergize each other to drive neuronal cells to survival. In contrast, the high levels of Ngb appear to play a pivotal role in sustaining the visual process acting as an O2 buffer and carrier.
In 2001, Cygb (21 kDa), also named histoglobin and stellate cell activation-associated protein, has been reported to be expressed in most vertebrate tissues and organs; the highest Cygb level occurs in the brain, eyes, liver, heart, and skeletal muscle [6, 7, 21, 30]. In the brain, Cygb is localized in piriform cortex, amygdala, hypothalamus (medial preoptic area, supra chiasmatic nucleus, lateral hypothalamus, ventromedial hypothalamic nucleus, and arcuate nucleus), hippocampus, reticular thalamic nucleus, habenular nuclei, laterodorsal tegmental nucleus, pedunculopontine tegmental nucleus, locus coeruleus, nucleus of the solitary tract, spinal trigeminal nucleus, and the dorsal raphe nucleus .
Members of the Cygb family display two peculiar features: (i) the occurrence of fairly long N- and C-terminal high-flexible extensions, possibly involved in lipid binding [84-87], and (ii) the bis-histidyl hexa-coordinated heme-Fe atom [84-86], the cleavage of the heme distal HisE7-Fe bond being pivotal for exogenous ligand binding [58, 86].
The reversible hexa- to penta-coordination transition of the heme-Fe-atom of Cygb and in turn the heme-Fe-atom-based reactivity are modulated by the redox state of the cell [58, 88] and lipid binding . Upon formation of the CysB2–CysE9 disulfide bond, the O2 affinity of the Cygb increases by about one order of magnitude (i.e., P50 changes from 2 to 0.2 mmHg; ref. 88). Moreover, the CysB2 and CysE9 residues have been reported to form intermolecular disulfide bridges stabilizing the hexa-coordinated Cygb homodimeric structure [85, 86]. In addition, lipid binding to Cygb induces the penta-coordination of the heme-Fe-atom, which facilitates lipid peroxidation and may assist the heme-protein dimerization . However, the oligomerization state of Cygb is an openly debated issue, Cygb behaving as a monomer in solution .
Cygb shows several properties, including (i) reversible binding of gaseous ligands (e.g., O2 and NO; ref. 87), (ii) NO dioxygenase activity , (iii) NO synthesis under anaerobic conditions , (iv) peroxidasic activity [6, 91], (v) lipid peroxidation , (vi) detoxification of reactive nitrogen and oxygen species [92, 93], and (vii) collagen metabolism [94, 95]. Accordingly, Cygb has been reported (i) to act as an O2 buffer and to facilitate O2 transport to mitochondria [6, 7], (ii) to play a role in NO signaling and involvement in sleep–wake cycling [30, 96], (iii) to protect cells against oxidative stress [97, 98], (iv) to play an antifibrotic activity especially on liver, involving apoptosis induction [94, 95, 99-103], (v) to modulate myogenic progenitor cell viability and muscle regeneration , (vi) to have bimodal tumor suppressor and oncogene functions [13, 32, 91, 98-108], and (vii) to serve as an independent predictive factor for prognosis of glioma patients . However, the low level of Cygb in several tissues and organs with the exception of the eye  casts doubts on some of these functions (e.g., O2 buffer and transport).
Cygb is upregulated on hypoxia and ischemia in most tissues and organs. The mechanism of induction of Cygb is regulated by the hypoxia-inducible factor 1α, a post-transcriptionally regulated transcription factor controlling several hypoxia-inducible genes [12, 21]. The up-regulation of Cygb results in reduced hypoxia-ischemia brain injury. In particular, Cygb upregulates the VEGF and superoxide dismutase, increases both the density and diameter of the microvessels, and inhibits the activation of caspase-2 and -3. The reduction of hypoxia-ischemia brain injury by Cygb may be due in part to antioxidant and antiapoptotic mechanisms and by promoting angiogenesis. Moreover, Cygb expression was associated with the improvement of long-term cognitive impairment .
Cygb could exert cell protection by catalyzing lipid peroxidation. Indeed, the products of Cygb-catalyzed lipid peroxidation would allow the cell to upregulate antioxidant defenses before extensive oxidative damage occurs or potentially to induce apoptosis if the balance of oxidative chemistry cannot be maintained . Remarkably, modified lipids formed by the redox activity of cytochrome c [110, 111], Hb, and Mb [112, 113] are known potent cell signaling molecules [114, 115]. Human Cygb is much more pro-oxidant than human Ngb, Hb, and Mb, suggesting that the upregulation of antioxidant defenses of the cell induced by lipid peroxidation is a prerequisite of human Cygb .
Human Cygb has been associated with some neurodegenerative disorders. In particular, Cygb localization in retinal layers with high oxygen demand and in the retinal ganglion cells, which are the primary cell populations affected by glaucoma and are very sensitive to ischemic diseases, suggests a role of this globin in sustaining the visual process in the eye. Remarkably, the upregulation of Cygb mRNA in glaucomatous mouse eyes has been suggested to display a pivotal role in retinal oxygen homeostasis enabling the retina to sustain long periods of ischemia [116, 117]. Moreover, human Cygb has been identified in the cytoplasmic eosinophilic inclusions of protoplasmic astrocytes of the neocortex, usually in the clinical setting of epilepsy and/or psychomotor retardation , and in the hyaline deposits of putaminal neurons and glia in a patient with hereditary ferritinopathy . The potential pathogenic importance of Cygb in neurodegenerative diseases may be related to the unbalanced protective effect of this globin against reactive nitrogen and oxygen species.
For a long time, Mb was believed to be expressed only in oxidative skeletal and cardiac muscle where it facilitates intracellular O2 diffusion and storage . In 2008, Mb was also detected in human medullo-myoblastoma and medulloblastoma [25, 28, 32]. Double immunopositivity for synaptophysin, neurofilament protein, and Mb in medullo-myoblastoma cells suggested that the neuroectodermal cells may undergo differentiation into rhabdomyoblasts . Moreover, although medulloblastoma demonstrating multipotent differentiation is rare, the diffuse expression of synaptophysin and neurofilament protein and the focal expression of the glial fibrillary acidic protein, the S-100 protein, desmin, and Mb support the idea that medulloblastoma originates from multipotent stem cells . However, the role of human Mb in health and disease is obscure; in fact, the expression of human Mb in the transgenic mouse brain, particularly in the hippocampus, cerebellum, and cerebral cortex, did not alter cell metabolism and morphology .
Over the last two decades, Hb α- and β-chains have been found either coexpressed in neurons, glial cells, alveolar cells, eye's lens, mesangial cells of the kidney, hepatocytes, and skeletal muscle [122-127] or non-coexpressed in endothelial cells and macrophages [128, 129]. However, only in 2013, Hb has been reported to retain the heterotetrameric structure in mouse mesencephalon as in blood; nevertheless, it remains to be determined whether both α- and β-chains can also coexist in the monomeric and/or homotetrameric forms . This suggests that α- and β-chains and tetrameric Hb functions are not exclusively restricted to the blood but may play multiple roles in health and disease.
In 2009, α- and β-chains of Hb were identified in the cytoplasm and nucleus of the large majority of A9 dopaminergic neurons of different strains of mice, rats, and human postmortem brains both as mRNA and protein [9, 10]. Hb α- and β-chains have also been found expressed in neurons of the cortex, hippocampus, cerebellum, and retina of rodent and human brains [10, 11, 27, 123, 130, 131]; however, the presence of Hb in glial cells is openly debated [9-11, 27, 132, 133].
The control of α- and β-globin genes expression during erythrocyte differentiation is perhaps the most studied example of transcriptional regulation in mammalian cells. Among the transcription factors crucial for globin genes transcription during erythrocytes differentiation, GATA family members have been found expressed in dopaminergic neurons of the substantia nigra suggesting their potential role in Hb transcription in the brain .
Recently, neuronal Hb expression in rat brain and retinal ganglion cells has been shown to be upregulated by erythropoietin, hypoxia, and ischemia as in erythrocytes [9, 11, 27, 131]. In addition, α- and β-globin transcripts are under positive control of HIF1 , IGF1 , and E2  levels.
In mouse cerebellar granule neurons, the Hb concentration is at least 50-fold lower than in the red blood cells . This observation has two important consequences: (i) there is no need for the presence of an ad hoc pathway for heme biosynthesis as in red blood cells; and (ii) it seems very unlikely that Hb may act as an oxygen carrier as in circulation. The function of Hb in the central nervous system remains unclear although several evidences suggest an involvement in mitochondria homeostasis [9, 10, 136, 137]. However, Hb induction by erythropoietin, hypoxia, and ischemia is believed to facilitate neuron oxygenation and may act as a neuroprotectant . Furthermore, the expression of α- and β-globins in the rat brain has been postulated to buffer the toxic heme released during clot resolution .
Mitochondrial dysfunction, oxidative stress, and iron metabolism are all well-established, crucial elements in neurodegeneration. Notably, α- and β-globin mRNAs have been shown to be strongly downregulated in animal models of depression and chronic stress , in the neurochemical model of Parkinson's disease , and in aging . Recently, it has been hypothesized that Hb may play a fundamental role in the association between cerebral vascular diseases and Huntington's disease (HD) or Alzheimer's disease [130, 140]. Interestingly, Hb β-chains have been reported to be interactors of huntingtin which mutation is, responsible for HD [141, 142]. Moreover, the reduced expression of Hb α- and β-chains has been observed in human neurons bearing granular or punctuates hyperphosphorylated tau deposits in the form of pretangles and tangles in the frontal cortex and hippocampus in HD and in pretangles in the argyrophilic grain disease. The loss of Hb α- and β-chains also occurs in all ballooned neurons in the amygdala in Alzheimer's disease and argyrophilic grain disease . In contrast, β-chains have been found to be upregulated in the mitochondria-enriched fractions and to control gray matter cortex of patients with multiple sclerosis . Furthermore, neuronal Hb may play a role in Parkinson's disease pathogenesis being linked to the alteration of mitochondrial oxidative phosphorylation, oxidative stress, and iron deposits in the A9 dopaminergic neurons . Of note, a functional polymorphism in the gene for the Hb-binding protein haptoglobin has been shown to influence susceptibility for idiopathic Parkinson's disease .
Fetal, embryonic, and adult Hb has been reported to be expressed in human glioblastoma multiforme, the most aggressive tumor among gliomas. In particular, human ε globin is predominantly expressed. It gradually decreases on increasing hypoxia, whereas α and γ globins concomitantly increase. The hypoxic upregulation of Hb expression in glioblastoma multiforme cells may be a part of a repertoire of active defense and adaptation mechanisms enabling these cells to acquire resistance to chemotherapy and radiotherapy. Therefore, new therapeutic strategies are required to interfere with Hb expression or function in glioblastoma multiforme cells [35, 36].
One possible approach to assess Hb as a potential modifier of neurodegenerative diseases might involve the study of β-thalassemia. As β-thalassemia minor has been shown to be protective against ischemic cerebrovascular accidents, advanced coronary artery disease, and myocardial infarctions, it has been hypothesized that β-thalassemia minor may be protective against the vascular pathogenesis of HD . However, there are no formal studies about the correlation between the β-thalassemia carrier state and other neurodegenerative disorders.
Lastly, the possible relationship between reduced neuronal Hb and anemia in the elderly and its impact on cognitive impairment and dementia could not be excluded [11, 31, 146-149].
Conclusion and Perspectives
Globins have been detected not only in erythroid cells and oxidative muscle but also in the nervous central system. Moreover, vertebrate nerve globins have been reported to be coexpressed not only in the brain and in the neuronal retina but also in different tissues and organs. This supports the view that these globins may play multiple roles in human health and disease.
The functions of mammalian nerve globins depend on their concentration; indeed, at high concentration (approximately 100 to 200 μM), they may facilitate O2 buffer and transport in tissues and organs (especially in the retina), whereas at low concentration (∼1 μM), they could display (pseudo-)enzymatic activities and could be part of signaling pathways.
Although the above-mentioned studies are inspiring in explaining mammalian nerve globin neuroprotection mechanisms, most of them are based on indirect or correlative experimental data. Thus, a better understanding of the molecular mechanisms at the root of biological functions of mammalian nerve globins bears fundamental and translational significance, with potential implications for the development of nerve globin-targeted therapeutics against stroke and neurological disorders.
The authors thank Dr. Marco Fiocchetti and Dr. Loris Leboffe for helpful discussions. This work was partially supported by a grant from Ministero dell'Istruzione, dell'Università e della Ricerca of Italy (PRIN 20109MXHMR_001 to P.A.).